ORIGINAL ARTICLE

The Potential Effect of Botulinum Toxin Type A on Human

Dermal Fibroblasts: An In Vitro Study
SANG-HA OH, MD,*§ YOUNG LEE, MD,†§ YOUNG-JOON SEO, MD,† JEUNG-HOON LEE, MD,† JUNG D.
YANG, MD,‡ HO Y. CHUNG, MD,‡ AND BYUNG C. CHO, MD‡

BACKGROUND The use of botulinum toxin type A (BoNT) continues to expand. Some physicians have noted
a face-lifting effect after intradermal injection of BoNT, although the effects are controversial.
OBJECTIVE To investigate the in vitro effects of BoNT on human dermal fibroblasts.
METHODS The proliferation and toxic effects of BoNT on human dermal fibroblasts were measured. To
understand the mechanism of BoNT on collagen production of fibroblasts, procollagen type I carboxy-
terminal peptide (PIP) was measured using enzyme-linked immunosorbent assay, and collagen production
was monitored using Western blotting. To examine the effect of BoNT on collagen degradation, we evaluated
matrix metalloproteinase (MMP) production using gelatin zymography.
RESULTS BoNT did not stimulate the proliferation of or show toxic effects on human dermal fibroblasts.
Levels of PIP increased significantly in fibroblasts grown in the presence of BoNT, and BoNT upregulated the
expression of type I collagen and decreased the production of some MMPs in fibroblasts that prevent collagen
degradation.
CONCLUSIONS This study shows interesting effects of BoNT on collagen production and degradation of
human dermal fibroblasts in vitro. This research provides the experimental background for using intradermal
BoNT injection for remodeling of dermal tissues in aged skin.
The authors have indicated no significant interest with commercial supporters.

I n the early 1980s, botulinum toxin type A

(BoNT) was first introduced for medical
indications such as strabismus and blepharo-
spasm.1,2 BoNT induces chemodenervation through
its action on presynaptic neurons, preventing the
release of acetylcholine and leading to functional
denervation of striated muscle for 2–6 months after
injection. This results in muscle fiber atrophy and
subsequent clinical flaccid paralysis.3 Consequently,
its cosmetic use in treating wrinkles induced by
muscle hyperactivity is widespread.4,5
Currently, many off-label cosmetic applications of
BoNT are under evaluation. Some physicians have
observed a face-lifting effect due to increased
collagen synthesis after intradermal injection of
BoNT to the mid and lower face,6,7 but the effect of
intradermal injection of BoNT is still controversial.
No experimental trials have been conducted to
support this observation, and the mechanism of
BoNT action intradermally has yet to be determined.
For these reasons, it was hypothesized that intra-
dermal injection with BoNT would stimulate

*
Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Chungnam National University,
Daejeon; †Department of Dermatology, Graduate School of Medicine, Chungnam National University, Daejeon;
‡
Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyungpook National University,
Daegu, South Korea
§
These authors contributed equally to this study.

© 2012 by the American Society for Dermatologic Surgery, Inc.  Published by Wiley Periodicals, Inc. 
ISSN: 1076-0512  Dermatol Surg 2012;1–6  DOI: 10.1111/j.1524-4725.2012.02504.x

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 EFFECTS OF BOTULINUM TOXIN ON FIBROBLASTS
remodeling of the extracellular matrix (ECM), a
process that requires activation of dermal fibroblasts
and is essential for the rejuvenation of aged skin.
The present study attempted to evaluate the
effectiveness of intradermal injection of BoNT.
Material and Methods
Cell Culture
Normal human skin samples were obtained from
circumcisions in accordance with the ethical com-
mittee approval process of Chungnam National
University Hospital. Specimens were sterilized in
70% ethanol, minced, and incubated in Dulbecco’s
modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum and antibiotics.
Dermal fibroblasts normally grew from the explants
after 5–7 days. Cells were starved of serum for
2 days and then treated with BoNT (Botox,
Allergan, Irvine, CA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide Assay
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) assay was performed to test
the cytotoxicity of BoNT to human dermal
fibroblasts. Human dermal fibroblasts were treated
with 0, 1, 2.5, or 5 U of BoNT for 6 hours. After
being washed twice with phosphate buffered saline,
cells received fresh medium. MTT solution was then
added to each well 4 hours before harvesting. The
medium was removed at the indicated time points,
and the resulting formazan crystals were solubilized
in dimethyl sulfoxide. The optical density was
determined at 540 nm using an enzyme-linked
immunosorbent assay (ELISA) reader. Measure-
ments were repeated five times.
[3H]-thymidine Incorporation Assay
3
[ H]-thymidine incorporation assay was performed
to investigate whether BoNT has the ability to
stimulate the proliferation of human dermal
fibroblasts. Human dermal fibroblasts were cultured
2 DERMATOLOGIC SURGERY
overnight in the absence of BoNT or with 1, 2.5, or
5 U of BoNT. Cultures were replenished with fresh
medium containing 1 lCi of [3H]thymidine
(Amersham, Little Chalfont, Buckinghamshire, UK).
Radioactivity in cell lysates was measured using liquid
scintillation. Measurements were repeated five times.
Procollagen Type I Carboxy-terminal Peptide
Assay
Because procollagens are synthesized as precursor
molecules of collagens, we measured the free
carboxy-terminal peptide of procollagen type 1 (PIP)
to quantify the amount of collagen molecules that
fibroblasts treated with BoNT synthesize. Cells were
serum-starved for 1 day and then further cultured in
serum-free DMEM with 0, 1, or 2.5 U of BoNT for
2 days. The level of PIP in the conditioned medium
was quantified using an ELISA kit (Takara Bio Inc.,
Shiga, Japan) using the manufacturer’s recom-
mended protocol.
Western Blot Analysis
The collagen production of fibroblasts after they
were treated with BoNT was analyzed using
Western blot analysis. Human dermal fibroblasts
were incubated with 0, 1, 2.5, or 5 U of BoNT for
2 days, and the expression levels of collagen, type I,
alpha 1 (COL1A1) and COL1A2 protein were
quantified using Western blot analysis. Total protein
was measured using a Bradford protein assay kit
(Bio-Rad Laboratories, Hercules, CA). Blots were
visualized using enhanced chemiluminescence
(iNtRON Biotechnology, Gyeonggi-do, Korea) and
quantified using an image analyzer (i-solution TM;
iMTechnology, Daejeon, Korea).
Gelatin Zymography (Collagen Degradation
Assay)
Gelatin zymography was used to detect matrix
metalloproteinase (MMP)-2 and MMP-9 and gela-
tinase secretion from human dermal fibroblasts after
BoNT treatment. Human dermal fibroblasts were
treated with 1, 2.5, or 5 U of BoNT and incubated
 OH ET AL

for 2 days. Medium from incubated dermal dose, although these differences were not significant
fibroblasts was loaded on sodium dodecyl sulfate (p > .05), which suggests that there is no notable
polyacrylamide gels. After electrophoresis, gels were cytotoxicity on human dermal fibroblasts of BoNT
stained using Coomassie brilliant blue. (Figure 1A).

Statistical Analysis Fibroblast Proliferation After BoNT Treatment

Statistical analysis was performed using SPSS ver-
sion 12.0 for Windows (SPSS, Inc., Chicago, IL). T-
tests and parametric analysis of variance (ANOVA)
with Scheffe post hoc test for multiple comparisons
were used for comparison between groups, with
p < .05 being regarded as significant. Data are
presented as mean values ± standard deviations.
Results
Cytotoxicity on Fibroblast After BoNT
Treatment
We performed an MTT assay to evaluate the
cytotoxicity of BoNT on human dermal fibroblasts.
The human dermal fibroblasts of the BoNT-treated
groups had slightly greater viability than control
fibroblasts that was inversely correlated with BoNT
We performed [3H]-thymidine incorporation assay
to determine whether BoNT promotes the prolifer-
ation of human dermal fibroblasts. As shown in
Figure 1B, the rate of proliferation was somewhat
higher in fibroblasts treated using 1 or 2.5 U of
BoNT and slightly lower in cells treated using 5 U of
BoNT, but none of these results were significant
(p > .05), indicating that BoNT does not promote
the proliferation of human dermal fibroblasts
(Figure 1B).
Collagen Production of Fibroblasts After BoNT
Treatment
PIP production, which indirectly reflects overall
collagen I levels, was examined to investigate
whether BoNT regulates collagen production in
cultured human dermal fibroblasts in vitro. PIP was

Figure 1. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of human dermal fibroblasts cultured for
1 day in the absence of botulinum toxin type A (BoNT) or with 1, 2.5, or 5 U of BoNT. Control fibroblasts appeared to
proliferate slightly less than those treated with BoNT; this effect was not significant (p > .05). Significant differences between
BoNT doses were not seen either. (B) [3H]thymidine uptake assay. Cells treated with BoNT (1 U or 2.5 U) appeared to have
proliferated slightly more, and those treated with 5 U BoNT appeared to have proliferated slightly less than controls, but
these differences were not significant (p > .05). Measurements were repeated five times.

2012 3
 EFFECTS OF BOTULINUM TOXIN ON FIBROBLASTS

Figure 2. The level of procollagen type I carboxy-terminal peptide (PIP) in conditioned medium was quantified using an
enzyme-linked immunosorbent assay kit. PIP was high in medium from cells treated with botulinum toxin type A after
36 hours (*p < .05 vs control). Measurements were repeated five times.

gradually elevated after 12 hours and rose gradually Discussion

over 48 hours in cells cultured in BoNT. PIP
Since the Food and Drug Administration (FDA)
production increased in a time- and BoNT dose–
granted approval for the use of BoNT in treatment
dependent manner (Figure 3). We also investigate
of glabellar rhytides in 2002,5 it has been used in a
the effect of BoNT on the expression of type I
variety of medical and nonsurgical treatments.3–7 It
collagen protein using Western analysis. Type I
appears that novel uses are being developed almost
collagen is the most abundant collagen component
every day, although at this point, most of them
of dermis and is assembled with pro-alpha collagen
remain “off label.” Eventually, the FDA may
chains encoded by COL1A1, which is the major
approve many of these new procedures, and the
component of type I collagen, and COL1A2.
potential for other new applications of BoNT
appears to be endless.5
According to Western blotting, expression of
COL1A1 was significantly greater in the BoNT-
Some physicians have noted a face-lifting effect
treated groups than in controls, and this effect was
after intradermal injection of BoNT to the mid and
dose-dependent (Figure 3A). Greater expression of
lower face. It has been claimed that greater colla-
COL1A2 was seen with exposure to 5 U of BoNT,
gen synthesis, lower sebum production, and
but cells treated with 1 or 2.5 U of BoNT showed
smaller facial pore size cause the effect.8,9 As
slightly less expression than in the control group
Kurzen and Schallreuter reported, the acetylcho-
(Figure 3B).
line receptor is not only present on neurons, but
can also be found on the surface of melanocytes,
MMP Production of Fibroblasts After BoNT
keratinocytes, and other dermal tissues. 10 One
Treatment
may reasonably suspect that an effect might be
We also examined the effects of BoNT on MMP-2 produced on adjacent tissue components after
and MMP-9 protein expression in cultured human BoNT injection, but what makes this argument
dermal fibroblasts. All BoNT-treated human dermal even more interesting is that percutaneous needle
fibroblasts showed less expression of pro-MMP-9 pricks themselves have been reported to create
and active-MMP-9 proteins in a dose-dependent multiple microbruises in the dermis and to initiate
manner than controls. Unlike MMP-9, MMP-2 a complex cascade of growth factors that eventu-
protein levels were not significantly different ally results in collagen production. Thus, intra-
between groups (Figure 4). dermal BoNT injection might not have any benefit

4 DERMATOLOGIC SURGERY
 OH ET AL

(A)

(B)

Figure 3. (A) Collagen, type I, alpha 1 (COL1A1) protein expression was quantified using Western blotting after 48 hours
of incubation with the indicated doses of botulinum toxin type A. COL1A1 expression was significantly higher in the
experimental groups than in the control group in a dose-dependent manner. (B) COL1A2 protein expression was
quantified using Western blotting. Although COL1A2 expression after treatment with 1 or 2.5 U of BoNT appeared to be
somewhat less than in controls, it increased with increasing doses of BoNT. Measurements were repeated three times.

main element of human skin. Collagen is responsible

Figure 4. Matrix metalloproteinase (MMP) activity in
cultured human dermal fibroblasts. Samples underwnet
gelatin zymography. The expression of pro-MMP-9 and
active-MMP-9 proteins decreased in botulinum toxin type A
–treated cells. MMP-2 protein level was not significantly
altered. Measurements were repeated five times; a repre-
sentative gel is shown here.
for maintaining the structural integrity of the skin by
joining cells together and to the extracellular matrix
(ECM). Fibroblasts secrete the precursors of
collagen: protocollagen types I and III. Aging brings
about a decrease in the number of skin fibroblasts, so
fibroblast proliferation might have antiaging effects.
Aging also brings a decrease in the number of skin
fibroblasts and, concomitantly, an increase in
MMPs, which catalyze the degradation of collagen.
MMPs are one family of structurally related
enzymes that have the collective ability to degrade
nearly all ECM components. In particular, the
gelatinases (MMP-2 and -9) are important effectors
of ECM remodeling.13

for facial rejuvenation, the purported benefits of
skin rejuvenation being due to the collagenesis
caused by the needle pricks. 11,12 Therefore, studies
of BoNT effects on collagenesis of human dermal
fibroblasts in vitro that exclude the effects of
needles must be designed.
Elastin, fibronectin, and collagen are the basic
elements of the fibrous component of the dermis,
and collagen is the most abundant of these and is the
We focused on the effect of BoNT on human
dermal fibroblasts, because the fibroblasts are the
main cellular component in dermis that secrete
collagen and MMPs. Before investigating the
ability of BoNT stimulating the production of
collagen synthesis by human dermal fibroblasts,
we examined the cytotoxicity of BoNT on human
dermal fibroblasts. BoNT did not show any
cytotoxicity or stimulate fibroblast proliferation
more than in controls.

2012 5
 EFFECTS OF BOTULINUM TOXIN ON FIBROBLASTS
PIP production, which reflects overall collagen
levels, was greater in BoNT-treated human dermal
fibroblasts. Consistent with PIP results, the pro-
alpha collagen chains alpha 1 (COL1A1), the major
component of type I collagen, and alpha 2
(COL1A2) protein levels were also higher after
BoNT treatment, suggesting that BoNT promotes
fibroblast activity, such as collagen production,
without increasing fibroblast proliferation and
cytotoxicity on fibroblasts.
Our study using a collagen degradation assay
(gelatin zymography) showed that BoNT decreased
the expression of pro-MMP-9 and active-MMP-9
proteins in human dermal fibroblasts. MMP-2 pro-
tein levels were not significantly changed, which
indicates that BoNT decreases collagen degradation.
In summary, we investigated the effects of BoNT on
collagen synthesis and MMPs production in human
dermal fibroblasts in vitro, which are essential for
the aging of skin. BoNT did not stimulate prolifer-
ation of fibroblasts but increased PIP and stimulated
the expression of type I collagen in human dermal
fibroblasts. BoNT also decreased production of
some forms of MMP, suggesting that BoNT has the
potential to promote remodeling of aged and photo-
aged skin and to be used as a method of facial
rejuvenation. Considering that there have been only
limited studies on clinical efficacy, further research
and clinical studies are required to investigate the
mechanisms of BoNT in dermal remodelling.
Acknowledgments This study was supported by
Grant from the Korea Healthcare Technology R&D
Project, Ministry of Health and Welfare, Republic of
Korea.
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Address correspondence and reprint requests to:
Sang-Ha Oh, MD, Chungnam National University
Hospital, 640 Daesa-dong, Jung-gu, Daejeon 301–721,
South Korea, or e-mail: djplastic@cnu.ac.kr